Critique: HRT Cafe

You can read about the spirit of the critique here.

This critique was written in February 2025 using the three HRT Cafe pages backed up here , here , and here . HRT Cafe publishes their guide here .

Summary

HRT Cafe’s two guides, “Injection Tutorial” and “Advanced Injection Tutorial”, are well intentioned, but miss the mark on several fronts. HRT Cafe is likely the most visible website on the internet that promotes material on how to homebrew. For this reason they should be held to a very high standard.

The major problems are the following:

1. The guides are overly concerned with controlling exposure to dust, but continuously fail to mention controlling for bacteria and viruses.
2. They promote the myth that steam based sterilization is possible for HRT, and they do not use any sources to support their claim. (Read more)
3. At several places they heat the solution up, either on a hot plate or in the autoclave, exposing benzyl alcohol to heat. This risks evaporation as well as toxic oxidation. (Read more)
4. They promote isopropyl alcohol 99% which is significantly less effective for disinfection than 70%. (Read more) (USP recommends 70%¹)
5. They downplay the importance of depyrogenation and claim that it’s “optional.” (USP says required²)
6. They recommend silicone stoppers as an alternative, which benzyl alcohol can evaporate out of. (Read more)
7. They recommend building a “moving air box” and further recommend a deeply inadequate filter for it. This is essentially a contraption that is exposing the entire brew process to contaminated forced air. Very unsafe.

For them to create stronger guides they would fix the issues above and introduce a strong concept of aseptic processing technique.

While the creator of this guide is clearly very intelligent and crafty, they are failing to properly research what they are claiming to be an expert on, and instead are making assumptions and claims based on what they think is probably correct. Someone in their position, at the middle of the DIY scene, should take more responsibility for the information they are passing along to the public.

The Guides

Experimenting with the last line of defense against vial contamination should not be taken lightly.

Hard agree. Don’t mess about with your Benzyl Alcohol. Unfortunately, the autoclaving found later in the HRT Cafe guide is risking converting the benzyl alcohol into benzaldehyde. More info about that here.

Filtering also doesn’t remove all bacteria …

While this is accurate, this article’s placement is misleading. This article is about sterilizing “Fresh Water” aka “Lake Water” or “River Water”. This is very different from working with components that are already relatively clean, like your API that’s straight from a lab, your BB and BA that are already sterile, or your oil that’s hopefully pharmaceutical grade and not overrun with microbes.

I recommend using steam-based terminal sterilization in combination with filtering and a preservative.

Steam sterilization is not terminal sterilization for non-aqueous solutions, and it doesn’t actually do what is being claimed. Non-aqueous solutions cannot be autoclaved. Further, as mentioned above, you should never heat up benzyl alcohol.

claims otherwise are not taking into account the amount of heat transfer which actually occurs.

As laid out in the sterilization theory page, the max heat that can be transferred in this scenario is the max temperature of the autoclave, typically 121°C, which is drastically below the recommended 160°C when steam isn’t making direct contact with the item to be sterilized. The steam cannot transfer more heat than actually exists in the system. The glass can’t transfer the moisture into the preparation, it needs heat, moisture, and pressure all in tandem. These three things together destroy microbes.

When it’s just heat, or just heat and pressure, then you’re dealing with dry heat sterilization, as the glass is keeping the steam from reaching the compounded preparation. Further, even if the steam could reach the non-aqueous preparation, it can only make contact with the top most layer of the preparation. Steam cannot penetrate oil. Every pharmaceutical compounding textbook I’ve checked agrees on this point: you cannot steam sterilize a non-aqueous solution.

According to Compounding Sterile Preparations, 4th edition, “For high-risk sterile compounding, documentation must be available to prove that methods of sterilization work properly.” Since we cannot find documentation stating that non-aqueous can be steam sterilized, and can only find information on the contrary, it seems safe to say that this doesn’t work. Let’s also not forget the risk autoclaving poses to the benzyl alcohol.

Again, from Compounding Sterile Preparations, 4th edition, “[Steam sterilization] is the preferred method to sterilize aqueous solutions and suspensions that have been verified to maintain their chemical and physical stability under the required conditions.” Reminder that our solution is non-aqueous and that it has not been proven to maintain chemical stability under those conditions.

Injection Tutorial

Instant Pot Pro Plus. Do NOT use a regular instant pot. It must be a 15 psi pressure cooker

While this isn’t bad advice, this isn’t strictly true. Regular Instant Pots such as the IP-DUO are proven to be effective if you run them long enough. More here. If you can manage it, do opt for the 15psi models though.

Isopropyl Alcohol 99%

Ahh! Isopropyl Alcohol (IPA) 99% is actually much less effective at killing microbes than IPA 70%. This is because the extra water helps penetrate the microbes that are for killing. It also prevents the IPA from drying out and evaporating before it’s had a chance to work. Always use IPA 70%¹. Read more .

Pre-sterilized Vials

These are good if you’re brewing a vial or two just for yourself. Any operation that’s larger than this and you should be sterilizing your own vials to ensure that it’s being done properly. It’s hard to trust that the people on the internet you’re getting “sterile” vials from is doing a good job with it.

Estradiol Enanthate 10mL 40 mg/mL

I understand this is the most popular size and concentration to make, but I will continue to state that it’s poor practice to make vials that are intended to last for about 80 weeks. More here.

Filling & Capping Vials

This should be done under a still air box at a minimum.

HRT Cat claims that bubble point values are readily available for MCT oil on the manufacturer website

This feels like a misunderstanding of how bubble points work. The max burst pressure or “bubble point” is provided by a manufacturer of filters (for high quality vendors, not ebay or amazon ones) as a PSI listing. This has nothing to do with what is being filtered. Not sure what’s being referenced here. I read through the HRT Cat page about this, and that claim is non-existent.

the specific liquid used greatly influences the test results

Right. You should always do the bubble point testing with your preparation, or a mimic of your preparation without the API.

Summary

All in all this is a unique approach to making vials. They are using pre-sterilized vials from a vendor to remove the need to sterilize glassware correctly. I personally think this introduces new risks, like not being able to trust a vendor, but that’s just me. I think if they changed two major things this guide would be a lot better and safer:

1. Remove the autoclaving.
2. Introduce a much higher standard of aseptic processing.

Caulk Gun filtering

Hey this is pretty cool. I recommend this technique on my page here. Kudos to HRT Cafe for figuring this one out.

Vacuum Syringe Filtering

While this is a cool concept, ultimately it’s flawed. Syringe filters are not designed to be subjected to a vacuum. I went ahead and built this, and I could be wrong here, but it looked to me like the housing of my filters were allowing a substantial amount of unsterile air to get introduced to the solution as it was passing out of the filter and into the vial. I did a full write up here.

Advanced Injections Homebrew Tutorial

I’m going to avoid repeating myself from the above critiques for the most part.

Pre-sterilized vials

This feels funny to me when this guide is advising people it costs around $1,000 to do. At this point, and if you’re brewing in this volume, you really should be sterilizing your own glass, but, to each their own. They offer links to buy off amazon, but do mention possible QC issues.

moving air box… 20” MERV 13 Air Filter

This is, drastically, the wrong filter. A MERV 13 filters traps “less than 75% of of air particles that are 0.3-1.0 micron in size” (source ). Laminar flow hoods are using HEPA filters, which are more like MERV 17. For comparison, our brew filter, which is the primary means of sterilization, controls for anything as small as 0.22 microns in size. What’s happening, then, is that this brew is being exposed to a box fan pushing contaminated air directly onto it.

While this will control for dust, as seems to be what they’re looking to accomplish, it is actually accelerating exposure to bacteria and viruses. This is a major red flag. The entity of the sterilization control in this guide is nullified by this mistake.

The reason laminar flow is the standard, and not a moving air box, is because with laminar flow we can predict where the air is going. It’s coming from the filter and it’s leaving in a straight line out. With something like the moving air box, we’re dealing with turbulent flow. There’s no way to predict what the air is doing. While, yes, it appears to be moving from the filter and out of the box, when you introduce variables like your hands, you stop being able to account for what the air is going to do. You could be creating air pockets and backflow and there would be no way to know. There’s a reason laminar flow is the standard and that people don’t build moving air boxes.

None of the injections you can purchase from other homebrewers, even the best, are completely controlled for pyrogens.

True. Maybe one day we’ll get there. I’d still prefer that all glassware is depyrogenated, not just sterilized. This is listed as a requirement in USP 797².

Dry Heat Depyrogenation (Optional)

I get why it’s being called optional, because we can’t control for pyrogens in other areas, but honestly, shouldn’t we still strive to control as many of them as we can? I feel like depyrogenating where we can is a no brainer. In my opinion, all guides should strive to eliminate as much contam as possible, and not cut corners like this.

You can purchase silicone stoppers which can also safely be heated to these temperatures too.

No, you cannot replace butyl rubber with silicone, don’t do this. Benzyl alcohol is proven to be able to evaporate out of silicone. Especially if you’re autoclaving it, and then it evaporates in the vial, and then it encounters the silicone, and can pass through it. (read more)

Cover the openings of the media bottle and the vials with aluminum foil

Nitpick, but in labs the recommendation is to double up the foil, for safety. You should also be sure to leave a corner on kind of loose so the water can evaporate out.

Depyrogenation is likely not a useful activity since we cannot combat pyrogens in the actual injection solution we are preparing.

This is borderline misinformation. Any single thing that we can depyrogenate, we should². It’s not optional. You remove as much contamination as possible. Always.

sideways to help prevent dust from getting in

I appreciate not wanting dust to get in, but, dust is not actually the problem here. The problem is microbes, which are invisible. Dust is a good signal that something went wrong, as if dust is present then microbes are also present. But the concern here is microbes. This line of thinking is repeated in several places all around these guides. I can’t tell if they don’t understand that they need to be controlling for microbes primarily or not.

Bring the water to a boil using the hot plate. Reduce the temperature and place the media bottle into the hot water bath for 10 minutes.

Okay this one is actually a big problem. The flash point of benzyl alcohol is 101°C/214°F. This is the temperature at which benzyl alcohol is evaporating so much that you can light the fumes on fire. Granted this is for a solution that is 100% BA. But regardless, getting our finished, filtered solution anywhere near this temperature is a really good way to evaporate the benzyl alcohol out of your preparation. Don’t do this.

Filling Vials

While they are controlling for dust, the existence of this “moving air box” fully exposes all vials processed in there to the bacteria that is in the air.

Terminal Sterilization

No, this is not terminal sterilization. See sterilization theory and the note above from the other guide they made. USP 797 says “Steam sterilization is not an option… if there is insufficient moisture to sterilize the CSP within the final, sealed, container closure system”². In the case of our specific CSP, there is no moisture inside the container.

Summary

1. The moving air box is introducing more contamination then it’s preventing
2. Heating the preparation for filtration and dispensing is compromising to the benzyl alcohol
3. Non-aqueous solutions cannot be terminally sterilized by steam. The only way to sterilize them is filtration and aseptic processing.
4. Heating the preparation, in all the various ways, risks oxidizing benzyl alcohol to benzaldehyde

This guide is unsafe, and is not recommended. If they switched to a still air box, removed the heating of solution with BA in it, removed the autoclave, and introduced strong aseptic technique, then it would be drastically improved.

I’m of the personal opinion that bottle top filtration is irresponsible without a laminar flow hood. This is because once the product is filtered into the bottle, that bottle has to get exposed to the air in order to dispense it. Even in a still air box, the air isn’t reliably clean enough to risk exposing the full batch to it. With the laminar flow hood, we know that the air that we’re exposing our preparation to is clean and that there is academic research backing up our methods.

Sources

View the library page for access to some PDFs.